Levinson, S. and Cagan, R. L. (2016). Drosophila cancer models identify functional differences between Ret fusions. Cell Rep 16: 3052-3061. PubMed ID: 27626672Summary:
Drosophila models of RET fusions CCDC6-RET and NCOA4-RET were generated and compared. Both RET fusions directed cells to migrate, delaminate, and undergo EMT, and both resulted in lethality when broadly expressed. In all phenotypes examined, NCOA4-RET was more severe than CCDC6-RET, mirroring their effects on patients. A functional screen against the Drosophila kinome and a library of cancer drugs found that CCDC6-RET and NCOA4-RET acted through different signaling networks and displayed distinct drug sensitivities. Combining data from the kinome and drug screens identified the WEE1 inhibitor AZD1775 plus the multi-kinase inhibitor sorafenib as a synergistic drug combination that is specific for NCOA4-RET. This work emphasizes the importance of identifying and tailoring a patient's treatment to their specific RET fusion isoform and identifies a multi-targeted therapy that may prove effective against tumors containing the NCOA4-RET fusion.

The RET (rearranged during transfection) receptor tyrosine kinase is the leading susceptibility locus for Hirschsprung's disease (HSCR), a congenital lack of neurons in the distal regions of the digestive tract. HSCR arises due to the abnormal migration and survival of enteric neuron precursors derived from the neural crest, which has been classified as a neurocristopathy. RET is also found to have a role in kidney development and in a subset of neuroendocrine cancers. The ligands for RET are members of the Glial cell line-derived neurotrophic factor (GDNF) family, which act by binding to a GDNF family receptor (GFR) to activate intracellular RET signaling, or the Neural cell adhesion molecule (NCAM). GDNF is an important component of vertebrate brain development and maintenance, with clinical relevance to Parkinson's disease (Myers, 2018)

GDNF ligands appeared with the emergence of jawed fish and GFRs underwent a gene expansion at the same time. This expansion coincides with the appearance of the neural crest, a distinguishing structure for vertebrates. Homologs of the RET and GFR receptors are present in invertebrates but are thought to function independently of each other, with GFRs operating in conjunction with Fas2/NCAM rather than with a soluble ligan. In Drosophila, the RET gene (Ret) is expressed by enteric neurons and epithelial progenitor cells of the adult midgut and is required for homeostasis of these populations (Perea, 2017). In the Drosophila embryo, Ret is expressed in the developing stomatogastric nervous system (SNS), a population of cells that delaminate and migrate along the developing gut to form the enteric nervous system (ENS), and Ret is also expressed in the Malpighian tubules, the fly equivalent of the kidney. A previous study observed expression of Gfrl promoter fragments in the developing SNS, suggesting that Ret and Gfrl might function together in this tissue (Hernandez, 2015). Using CRISPR this study generated Drosophila Ret alleles and found defects in embryonic SNS formation and larval SNS function. These phenotypes led identification of the novel TGFβ family member Maverick (Mav) as an invertebrate GFR/Ret ligand and a candidate for the ancestor of GDNF. The results reveal remarkable similarities in the signaling mechanisms used to generate the insect SNS and the vertebrate ENS (Myers, 2018)

This study describes the effects of mutating the Ret gene in Drosophila and uncovered an evolutionarily conserved role in the development of the ENS. The incorrect positioning of SNS cells in the Drosophila embryo resembles hypoganglionic ENS phenotypes seen when RET is mutated in vertebrates. In HSCR, the most distal nerves of the digestive tract are affected. Likewise, in Ret mutant larvae the most distal nerves of the SNS, located on the midgut, have an altered anatomy and the larvae show defects in food ingestion. The phenotype resembles the neurotrophic effects of decreased serotonin or CNS dopamine signaling during midgut nerve formation, which also leads to increased axon branching and decreased feeding (Myers, 2018)

Although defects are visible in the embryonic SNS, there appear to be two separate lethal phases. Some first instar larvae display feeding defects and die. This is particularly evident in the original alleles that carry the background recessive lethal mutation, and the possibility is being investigated that the background lethal mutation specifically enhances the Ret mutations. Subsequent larval feeding defects often do not emerge until 2-4 days after hatching. Larvae with food in their guts can be observed foraging, suggesting that the larvae have problems with food ingestion. This is supported by observations of mutant larvae with food throughout their midguts, but with peristaltic defects in the anterior midgut. Initially a neurodegenerative defect similar to Wallerian degeneration was expected, but the axon defect was not suppressed by reducing dSarm activity. A model is currently favored in which initial SNS defects are amplified as the larva dramatically increases its mass several hundred fold. To keep pace with the expanding midgut, Ret may be required to promote axon growth, guidance, or be fulfilling a pro-synaptic role. These functions have been observed for RET and GDNF (Myers, 2018)

The midgut axon phenotype resembles defasciculation of the nerves and Gfrl genetically interacts with the fasciculation molecule Fas2, so Ret/Gfrl could potentially be modulating fasciculation as has been observed for other signaling systems. Alternatively, defasciculation may be a consequence of growth cones searching for sources of ligand, as proposed for Netrin and Bolwig's nerve. Decreased midgut innervation and function may provide negative feedback to upstream gut signaling, decreasing the ability to pass food through the pharynx and esophagus. The midgut axons may also be required to maintain communication with downstream enteroendocrine cells. An alternative hypothesis raised by the similarity of the Ret and Pink1 phenotypes is that the midgut neurons are running out of energy due to mitochondrial dysfunction (Myers, 2018).

This analysis enabled identification of the divergent TGFβ Mav as the elusive ligand for Drosophila Ret. The expression pattern of mav is consistent with a role in embryonic SNS development. Although the Mav ligand is concentrated in certain regions of the foregut and may create localized gradients, the broad expression pattern suggests that the Ret/Gfrl signaling pathway could be permissive rather than instructive during SNS precursor migration. Embryonic Ret signaling could primarily transduce a neurotrophic signal, and apoptosis has been observed in the migrating SNS precursors. In vertebrates, models in which GDNF/Ret signaling promotes proliferation rather than cell migration have been proposed to explain development of the nervous system. Experiments are underway to distinguish between these models in the fly. Although Gfrl expression has not yet been observed in the SNS, Gfrl could be acting in a soluble form or in trans. Gfrl promoter fragments continue to drive expression in the anterior midgut of the larvae in support of the trans model. Despite extensive sequence divergence in the extracellular domain of Ret, domain differences in GFRs and low homology of Mav to the GDNF family, the molecular logic of the protein complex appears preserved. In vertebrates, RET and GFR form a preassembled complex, and GDNF binds GFR to activate RET. Molecular data are strikingly similar, as this study found that Drosophila Ret and Gfrl can functionally interact in the absence of Mav, and that Mav interacts strongly with Gfrl, but only very weakly with Ret. In flies, Mav modulates synapse formation at the neuromuscular junction of body wall muscles. Ret is not expressed in body wall muscles , and Mav is likely to be signaling through activin/BMP type 1 receptors. A Mav homolog, Panda, has been found in the sea urchin Paracentrotus lividus, where it plays a role in dorsoventral axis formation and is also likely to be signaling through type 1 receptors. Mav and Panda both lack a key leucine residue, so their binding to type 1 receptors might be weaker than other ligands. Candidate Ret and Mav homologs have been found in Strongylocentrotus purpuratus, suggesting that Mav homologs might interact with both type 1 and Ret receptors in sea urchins (Myers, 2018).

Ret exhibits highly dynamic mRNA expression in the embryo. Ret is also expressed in adult midgut precursors at an earlier stage in development, as well as in discrete cells in the CNS, PNS and Malpighian tubules. mav mRNA is expressed weakly in the foregut primordium and at later stages in the pharynx, esophagus and proventriculus. Analysis of an epitope-tagged Mav expressed at endogenous levels indicates strong expression in the epithelial region from which the SNS precursor clusters delaminate and expansion to match the pattern of the mRNA, becoming concentrated near the sites at which the SNS neurons stop migrating (junction of the pharynx and esophagus, proventriculus). mav is also expressed in the epidermis and visceral mesoderm. Apart from promoter fragments driving reporters, Gfrl expression has not been observed in the SNS. Gfrl could therefore be expressed at low levels, or the protein might be acting in trans or in a soluble form. Gfrl promoter fragments continue to drive expression in the anterior midgut of the larvae (Myers, 2018).

Despite promiscuity in binding between TGFβ and their receptors in vertebrates, GDNF family members have not been reported to bind BMP/TGFβ receptors, suggesting that the ability to interact with more than one receptor was lost during evolution. The GDNF family of ligands, including GDNF, Neurturin, Artemin and Persephin, all appeared when fish gained jaws, as homologs cannot be identified in the published Agnatha sequences. GDNF ligands are distinguished by a highly conserved DLGLGY motif, part of one of two fingers that mediate binding to GFRα. This motif is not present in Mav or Panda. The change may have increased affinity or specificity for GFRs and additional changes might have prevented crosstalk with Activin/BMP type 1 receptors. Mav and Panda are similar to GDF-15, a TGFβ placed in the subfamily containing GDNF. GDF-15 is an inflammatory cytokine, and although it activates SMAD signaling, GDF-15 does not have an identified receptor. GDF-15 has GDNF-like neurotrophic activity for dopaminergic neurons, so it would be interesting to test GDF-15 for binding to GFRs (Myers, 2018).

The limited sequence data available suggest a model in which a divergent TGFβ acquired an ability to bind GFRs and activate Ret, which was followed by extensive co-evolution of the extracellular components. However, the downstream signaling pathways appear to be conserved, so the Ret SNS phenotypes open the door to invertebrate genetic analysis of this clinically important signaling pathway. Particularly exciting is the possibility of functional suppressor screens to identify mutations that could compensate for a lack of Ret signaling. Drosophila has already been used to identify genetic modifiers and a candidate drug to counteract oncogenic Ret signaling (Myers, 2018).

It is concluded Ret has an evolutionarily conserved role in the formation and function of the ENS. The GDNF signaling pathway has its origins in TGFβ signaling (Myers, 2018).